Aggregation Of Class A G Protein-Coupled Receptor And The Association With Downstream Signaling

GPCRs?G protein-coupled receptors?aggregation are characterized by special structures and functions in signaling transduction.It's well studied that Class C GPCRs could form constitutive dimers which are essential for their biological functions.However,the roles of Class A GPCRs aggregation in the process of signaling transduction remains unclear.In this article,we focused on the two typical Class A GPCRs,CCR3 and ?2-adrenergic receptor.The aggregation of the two receptors on the living cell membrane and the association with downstream signaling transduction protein?-arrestin and G protein heterotrimeric complexes was systematically studied.We also investigated the interaction between?-arrestin and G protein both in living cells and in vitro.Firstly,single-molecule imaging technique was applied to determine the stoichiometry of the two Class A GPCRs on the living cell membrane.The results showed that the two types of Class A GPCRs mainly existed as monomers under nearly physiological conditions and the aggregation status of two receptors was strongly correlated with their expression level.The aggregation status of the two Class A GPCRs could be regulated by agonists binding.The effect of regulation by agonists binding is correlated with expression level of receptors and pharmacology of different agonists.Our data supported the view that full agonists showed slight effect on regulation of Class A GPCRs aggregation.Full agonist isoproterenol showed no effect on the aggregation of ?2-AR.CCL11 and CCL24 could induce CCR3 aggregation at only high concentrations and the effect was weaker than biased agonists.Biased agonists could strongly induce Class A GPCRs aggregation.Biased ligand carvedilol and ICI-118,551could promote ?2-AR aggregation obviously,CCL8 also shows strong effect on CCR3 aggregation.Antagonists showed no obvious effect on Class A GPCRs aggregation.Secondly,we applied single-molecule imaging to visualize aggregation of two Class A receptors upon agonists binding using siRNA-mediated knock-down of ?-arrestin.The results showed that aggregation induced by carvedilol and ICI-118,551 was significantly inhibited.Similarly,the aggregation status of CCR3 induced by CCL8 also markedly decreased.The results showed that?-arrestin might be involved in the regulation of aggregation of two Class A GPCRs induced by biased agonists.The aggregation of CCR3 induced by CCL11 and CCL24 was inhibited using PTX pretreatment to uncouple G protein which suggested that G protein might be involved in CCR3 aggregation with CCL11 and CCL24 stimulation.The data above revealed that the aggregation status of the two type A GPCR on the cell membrane is closely related to the key protein in downstream signal transduction,?-arrestin or G-protein.In order to further explore the interaction between CCR3 and ?-arrestin on the living cells,?-arrestin-EGFP translocation was visualized in CCR3 stablely transfected cells with CCL11binding by confocal fluorescence microscope.The results showed that?-arrestin translocation in CCR3 stablely cells could be effected by CCL11 stimulation for 30 min.FRET signal with different contransfection ratios of CCR3-EYFP and ?-arrestin-ECFP was determined.The results suggested that?-arrestin could consititutively associate with CCR3.The FRET ratios decreased by CCL11 stimulation which indicated that this constitutive interaction between ?-arrestin and CCR3 could be modulated by CCL11.The migration of CCR3 stably transfected cells induced by CCL11 was inhibited obviously with siRNA-?-arrestin knockdown.The data indicated that?-arrestin might play an important role in CCL11 triggered CCR3 migration.Finally,we established a method of purification of?-arrestin in a prokaryotic system.The optimalizing heterogenous expression conditions was determined by optimizing the induction time and induction phase.?-arrestin and?-arrestin?R169E?mutant were produced with high purity using the methods above.The results of mass spectrometry analysis and circular dichroism?CD?spectrum suggested that the produced ?-arrestin?R169E?mutant had correct molecular mass and secondary structure.Quartz crystal microbalance technique was applied to determine the interaction of ?-arrestin?R169E?and CCR3.The result suggested that there is also an obvious interaction between ?-arrestin?R169E?and CCR3 in vitro and the equilibrium dissociation constant?K_D? between CCR3 and ?-arrestin?R169E?was 1.35×10^-7M.